Method for the purification of alphavirus replicon particles

ABSTRACT

Methods of production and purification for viruses and virus-derived vectors, including those related to alphaviruses, are disclosed. in one aspect, methods of purification that subject alphavirus replicon particle preparations to one or more steps of chromatographic purification, such as using an ion exchange resin, are provided. Also disclosed are methods of characterizing alphavirus replicon particles and utilizing these materials for vaccines and gene-based therapeutics.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/881,575 filed Jun. 29,2004, which is a divisional application of Ser. No. 09/872,086, filedMay 31, 2001 (now U.S. Pat. No. 6,767,699, issued Jul. 27, 2004), whichclaims the benefit of Ser. No. 60/208,376 filed on May 31, 2000. Each ofthese applications is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the purification of virusesand virus-derived vectors, including those related to alphaviruses, frombiological and chemical preparations. In particular, this inventionrelates to methods of purification of such viruses and vectors frompreparations by subjecting the preparation to chromatographicpurification using an ion exchange resin or combination of an ionexchange resin step and another chromatographic process step such assize exclusion or affinity chromatography. The method provides purifiedviruses and vectors for use as effective vaccines and therapeutics.Moreover related methods for quantifying replicon vector preparationsand verifying the replication incompetency of purified vectors areprovided.

BACKGROUND OF THE INVENTION

Alphaviruses comprise a set of genetically, structurally, andserologically related arthropod-borne viruses of the Togaviridae family.Twenty-six known viruses and virus subtypes have been classified withinthe alphavirus genus, including, Sindbis virus, Semliki Forest virus,Ross River virus, and Venezuelan equine encephalitis virus.

Sindbis virus is the prototype member of the Alphavirus genus of theTogaviridae family. Its replication strategy has been well characterizedin a variety of cultured cells and serves as a well-accepted model forother alphaviruses. Briefly, the genome of Sindbis virus (like otheralphaviruses) is an approximately 12 kb single-stranded positive-senseRNA molecule which is capped and polyadenylated, and contained within avirus-encoded capsid protein shell. The nucleocapsid is furthersurrounded by a host-derived lipid envelope into which twoviral-specific glycoproteins, E1 and E2, are inserted and anchored tothe nucleocapsid. Certain alphaviruses (e.g., SFV) also maintain anadditional protein, E3, which is a cleavage product of the E2 precursorprotein, PE2.

After virus particle adsorption to target cells, penetration, anduncoating of the nucleocapsid to release viral genomic RNA into thecytoplasm, the replicative process occurs via four alphaviralnonstructural proteins (nsPs), translated from the 5′ two-thirds of theviral genome. Synthesis of a full-length negative strand RNA, in turn,provides template for the synthesis of additional positive strandgenomic RNA and an abundantly expressed 26S subgenomic RNA, initiatedinternally at the junction region promoter. The alphavirus structuralproteins (sPs) are translated from the subgenomic 26S RNA, whichrepresents the 3′ one-third of the genome, and like the nsPs, areprocessed post-translationally into the individual proteins.

Several members of the alphavirus genus are being developed as“replicon” expression vectors for use as vaccines and therapeutics.Replicon vectors may be utilized in several formats, including DNA, RNA,and recombinant vector particles. Such replicon vectors have beenderived from alphaviruses that include, for example, Sindbis virus(Xiong et al., Science 243:1188-1191, 1989; Dubensky et al., J. Virol.70:508-519, 1996; Hariharan et al., J. Virol. 72:950-958, 1988; Polo etal., PNAS 96:4598-4603, 1999), Semliki Forest virus (Liljestrom,Bio/Technology 9:1356-1361, 1991; Berglund et al., Nat. Biotech.16:562-565, 1998), and Venezuelan equine encephalitis virus (Pushko etal., Virology 239:389-401, 1997). A wide body of literature has nowdemonstrated efficacy of such replicon vectors for applications such asvaccines (see for example, Dubensky et al., ibid; Berglund et al., ibid;Hariharan et al., ibid, Pushko et al., ibid; Polo et al., ibid; Davis etal., J Virol. 74:371-378, 2000; Schlesinger and Dubensky, Curr Opin.Biotechnol. 10:434-439, 1999; Berglund et al., Vaccine 17:497-507,1999).

Because of their configuration, vector replicons do not express thealphavirus structural proteins necessary for packaging into recombinantalphavirus particles (replicon particles). Thus, to generate repliconparticles, these proteins must be provided in trans. Packaging may beaccomplished by a variety of methods, including transient approachessuch as co-transfection of in vitro transcribed replicon and defectivehelper RNA(s) (Liljestrom, Bio/Technology 9:1356-1361, 1991; Bredenbeeket al., J. Virol. 67:6439-6446, 1993; Frolov et al., J. Virol.71:2819-2829, 1997; Pushko et al., Virology 239:389-401, 1997; U.S. Pat.Nos. 5,789,245 and 5,842,723) or plasmid DNA-based replicon anddefective helper constructs (Dubensky et al., J. Virol. 70:508-519,1996), as well as introduction of alphavirus replicons into stablepackaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999; U.S.Pat. Nos. 5,789,245, 5,842,723, and 6,015,694; PCT publications WO9738087 and WO 9918226).

Alphavirus replicon particles produced using any of the abovemethodologies subsequently are harvested in the cell culturesupernatants. The replicon particles then may be concentrated andpartially purified using one of several published approaches, includingpolyethylene glycol (PEG) precipitation, ultracentrifugation, orCellufine sulfate™ ion exchange chromatography. Unfortunately, thesemethods do not remove a sufficient level of non-alphavirus derivedprotein contaminants, are not scalable, or are costly, and therefore arelikely not amenable for commercial manufacture necessary of vaccine andtherapeutic products.

The present invention provides methods of production and purificationwith utility for the large-scale manufacture of alphavirus repliconparticles. Also disclosed are novel methods for quantitating vectorparticles in a preparation and determining the presence or absence ofcontaminating replication-competent virus in a preparation. Additionalmethods are provided for detecting the presence of packaged helper RNAsin a preparation of replicon particles. Alphavirus particles producedand characterized according to the methods described herein may be usedfor a variety of applications, including for example, vaccines and genetherapy.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides methods of production andpurification for alphavirus replicon particles. Such replicon particlesmay be derived from a wide variety of alphaviruses (e.g., Semliki Forestvirus, Ross River virus, Venezuelan equine encephalitis virus, Sindbisvirus), and are designed to express a variety of heterologous proteins(e.g., antigens, immunostimulatory proteins, therapeutic proteins).

Within one aspect of the invention, a method of purifying alphavirusreplicon particles is provided. Purification is achieved by firstcontacting a preparation containing alphavirus replicon particles withan ion exchange resin, under conditions and for a time sufficient tobind to the resin. Next, the portion of the preparation which is notbound to the ion exchange resin is removed from the ion exchange resin,and then the bound alphavirus replicon particles are eluted from the ionexchange resin and recovered. In one embodiment, the ion exchange resinis a tentacle ion exchange resin. In another embodiment, the tentacleion exchange resin is a cationic exchange resin. In yet anotherembodiment, the tentacle ion exchange resin is an anionic exchangeresin.

Within another aspect of the invention, a method of purification foralphavirus replicon particles is provided, comprising at least twochromatographic purification steps. The chromatographic purificationsteps are selected from the group consisting of ion exchangechromatography, size exclusion chromatography, hydrophobic interactionchromatography, and affinity chromatography. In one preferredembodiment, purification is performed using a first step of ion exchangechromatography and a second step of size exclusion chromatography.

Within another aspect of the invention, a method of producing alphavirusreplicon particles is provided. Alphavirus packaging cells are infectedwith a seed stock of alphavirus replicon particles and then incubated ina bioreactor, under conditions and for a time sufficient to permit theproduction of alphavirus replicon particles. Next the culturesupernatants containing the replicon particles are harvested. In oneembodiment, the bioreactor is an external component bioreactor. Inanother embodiment the bioreactor is a suspension culture bioreactor.

Within another aspect of the invention, a method of producing alphavirusreplicon particles is provided. Alphavirus packaging cells aretransfected with a DNA-based alphavirus replicon or eukaryotic layeredvector initiation system and then incubated in a bioreactor, underconditions and for a time sufficient to permit the production ofalphavirus replicon particles. Next the culture supernatants containingthe replicon particles are harvested.

Within another aspect of the invention, a method of producing alphavirusreplicon particles is provided wherein alphavirus packaging cells aretransfected with an alphavirus RNA vector replicon transcribed in vitroand then incubated in a bioreactor, under conditions and for a timesufficient to permit the production of alphavirus replicon particles.Next the culture supernatants containing the replicon particles areharvested.

Within other aspects of the invention, methods of generating alphavirusreplicon particles for use in vaccine or therapeutic applications areprovided. Replicon particles are produced in packaging cell lines andpurified by a chromatographic purification process as described above.In preferred embodiments, the chromatographic purification processincludes a step of ion exchange chromatography using a tentacle ionexchange resin.

Within yet other aspects of the present invention, a preparation ofalphavirus replicon particles is provided, wherein the preparation ofreplicon particles is purified by a chromatographic purification processas described above. In preferred embodiments, the chromatographicpurification process includes a step of ion exchange chromatographyusing a tentacle ion exchange resin.

Within a related aspect, a vaccine or immunogenic composition comprisinga preparation of alphavirus replicon particles purified by achromatographic purification process as described above is provided. Thepreparation of replicon particles being capable of expressing an antigenderived from a pathogenic agent. In preferred embodiments, thechromatographic purification process includes a step of ion exchangechromatography using a tentacle ion exchange resin. In one embodiment,the antigen is derived from a tumor cell. In another embodiment, theantigen is derived from an infectious agent (e.g., virus, bacteria,fungus, and parasite). In preferred embodiments, the antigen is derivedfrom HIV (e.g. gag, gp120, gp140, gp160, pol, rev, tat, and nef) or HCV(e.g. C, E1, E2, NS3, NS4, and NS5).

Within yet other related aspects, methods for stimulating an immuneresponse within a warm-blooded animal, comprising the step ofadministering to a warm-blooded animal a preparation of alphavirusreplicon particles purified by a chromatographic purification process asdescribed above are provided, the preparation of replicon particlesbeing capable of expressing an antigen derived from a pathogenic agent.In preferred embodiments, the chromatographic purification processincludes a step of ion exchange chromatography using a tentacle ionexchange resin. In one embodiment, the antigen is derived from a tumorcell. In another embodiment, the antigen is derived from an infectiousagent (e.g., virus, bacteria, fungus, parasite). In preferredembodiments, the antigen is derived from HIV or HCV.

Within yet other related aspects, methods for stimulating an immuneresponse within a warm-blooded animal, comprising the step ofadministering to a warm-blooded animal a preparation of alphavirusreplicon particles purified by a chromatographic purification process asdescribed above are provided, the preparation of replicon particlesbeing capable of expressing a lymphokine, cytokine, or chemokine. Inpreferred embodiments, the chromatographic purification process includesa step of ion exchange chromatography using a tentacle ion exchangeresin. In one embodiment, the lymphokine, cytokine or chemokine isselected from the group consisting of IL-2, IL-10, IL-12, gammainterferon, GM-CSF, MIP3α, MIP3β, and SLC.

Still other embodiments of the present invention provide for techniquesused to establish vector particle preparation safety and potency. Oneimportant aspect of vector particle safety is that the preparation befree of contaminating replication-competent alphaviral particles. Thepackaging cell lines used to produce the vector particles of the presentinvention contain at least three separate nucleic acid sources used toproduce the vector particles of the present invention. One nucleic acidsource contains nonstructural viral proteins and a gene of interest,another contains genes encoding for structural proteins and a thirdencodes for structural proteins not present in any other nucleic acidsource. Therefore, contaminating replication-competent alphaviralparticles can only arise if a minimum of two recombination events occur.

In one embodiment a preparation of replicon particles free fromdetectable contaminating replication-competent alphaviral particles isassured using polymerase chain reaction (PCR) techniques wherein anucleic acid substrate suitable for detecting multiple recombinationevents is provided. The substrate is derived from a population ofalphavirus replicon particles and the nucleic acid substrate is reactedwith at least one first reaction mixture comprising an oligonucleotidecomplementary to an alphavirus nonstructural protein gene and anoligonucleotide complementary to an alphavirus structural protein gene.The structural protein gene is either a capsid protein gene or anon-capsid structural protein gene. Suitable reaction conditions andtime are provided to permit amplification of the nucleic acid substrateand the formation of a first reaction product. Next, the first reactionproduct is reacted with a second reaction mixture containing anoligonucleotide complementary to an alphavirus capsid protein gene andan oligonucleotide complementary to a non-capsid alphavirus structuralprotein gene. Suitable reaction conditions and time are provided topermit amplification of the nucleic acid substrate and the formation ofa second reaction product. After the first and second reactions arecomplete, the presence or absence of the second reaction product isestablished.

In another embodiment of the present invention multiple recombinationevents are detected by providing a nucleic acid substrate suitable fordetecting multiple recombination events, the substrate being derivedfrom a population of alphavirus replicon particles. Then reacting thenucleic acid substrate with a first reaction mixture comprising anoligonucleotide complementary to an alphavirus nonstructural proteingene and an oligonucleotide complementary to an alphavirus capsidprotein gene. Conditions suitable and for a time sufficient to permitamplification of the nucleic acid substrate to form a first reactionproduct are provided. Next, the first reaction product is reacted with asecond reaction mixture comprising an oligonucleotide complementary toan alphavirus capsid protein gene and an oligonucleotide complementaryto a non-capsid alphavirus structural protein gene. Again, underconditions suitable and for a time sufficient to permit amplification ofthe nucleic acid template to form a second reaction product. Finally,determining the presence or absence of the second reaction product

In yet another embodiment a method for detecting multiple recombinationevents is provided comprising providing a nucleic acid substratesuitable for detecting multiple recombination events. The substrate isderived from a population of alphavirus replicon particles and thenreacting the nucleic acid substrate with a first reaction mixturecomprising an oligonucleotide complementary to an alphavirusnonstructural protein gene and an oligonucleotide complementary to anon-capsid alphavirus structural protein gene. Suitable reactionconditions and time are provided to permit amplification of the nucleicacid substrate to form a first reaction product. Next the first reactionproduct is reacted with a second reaction mixture comprising anoligonucleotide complementary to an alphavirus capsid protein gene andan oligonucleotide complementary to a non-capsid alphavirus structuralprotein gene. After a suitable incubation time, the presence or absenceof the second reaction product is determined.

In one preferred embodiment, at least two of the above methods fordetecting multiple recombination events are performed using the samenucleic acid substrate derived from a population of alphavirus repliconparticles.

In another embodiment of the present invention replicon particlepreparation potency is quantified. In this embodiment, methods areprovided for quantitating or “titering” replication incompetent RNAvirus vector particles in a sample. The methods comprising providing apopulation of packaging cells, contacting the packaging cells with thesample under conditions suitable and for a time sufficient for the cellsto be infected with replication-incompetent virus vector particles. Thenincubating the infected packaging cells under conditions suitable andfor a time sufficient for production of virus vector particles andenumerating the number of resulting plaques.

These and other aspects and embodiments of the invention Will becomeevident upon reference to the following detailed description andattached figures. In addition, various references are set forth hereinthat describe in more detail certain procedures or compositions (e.g.,plasmids, sequences, etc.), and are therefore incorporated by referencein their entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of an alphavirus replicon packagingcell line with a split structural protein gene expression cassetteconfiguration.

FIG. 2 is a graph showing the production of alphavirus repliconparticles using packaging cell line #15-25, in a 10 layer Cell Factory.

FIG. 3 is a schematic illustration of a CellCube™ bioreactor system

FIG. 4 is a graph showing the scale-up production of 90 liters ofalphavirus replicon particles using early generation packaging celllines in a 100-stack CellCube™ system.

FIG. 5 is a graph comparing the purification of alphavirus repliconparticles using two different single-step methods of ion exchangechromatography.

FIG. 6 are Coomassie stained protein gels comparing the purification ofalphavirus replicon particles using two different single-step methods ofion exchange chromatography.

FIG. 7 is a graph showing the purification of alphavirus repliconparticles using the tentacle cationic exchange resin s-Fractogel®.

FIG. 8 are Coomassie and silver stained protein gels showing thepurification of alphavirus replicon particles using a two stepchromatographic process.

FIG. 9 is a graph showing the induction of HIV gag antigen specific Tcells using alphavirus replicon particles subjected to PEG precipitationor single-step Fractogel chromatographic purification or two-stepFractogel/S400 chromatographic purification.

FIG. 10 is a graph showing the anti-tumor effect of SIN alphavirusreplicon particles expressing IL2 in a CT26 colon carcinoma model, ascompared to recombinant IL-2 protein or SIN replicon particlesexpressing GFP reporter.

FIG. 11 is a graph showing the use of bDNA assay for detection andquantitation of replicon RNA in a preparation of alphavirus repliconparticles as a means to determine titer.

FIG. 12 is a schematic illustration of a method for detection ofmultiple recombination events using nucleic acid amplification todetermine the presence or absence of contaminating replication-competentvirus in a preparation of alphavirus replicon particles

DEFINITION OF TERMS

The following terms are used throughout the specification. Unlessotherwise indicated, these terms are defined as follows:

“Alphavirus RNA vector replicon”, “RNA vector replicon” and “replicon”refers to an RNA molecule which is capable of directing its ownamplification or self-replication in vivo, within a target cell. Todirect its own amplification, the RNA molecule should encode thepolymerase(s) necessary to catalyze RNA amplification (e.g., nsP1, nsP2,nsP3, nsP4) and contain cis RNA sequences required for replication whichare recognized and utilized by the encoded polymerase(s). An alphavirusRNA vector replicon should contain the following ordered elements: 5′viral sequences required in cis for replication (also referred to as 5′CSE), sequences which, when expressed, code for biologically activealphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), 3′viral sequences required in cis for replication (also referred to as 3′CSE), and a polyadenylate tract. The alphavirus RNA vector replicon alsomay contain a viral subgenomic “junction region” promoter, which may, incertain embodiments, be modified in order to increase or reduce viraltranscription of the subgenomic fragment, and heterologous sequence(s)to be expressed.

“Recombinant Alphavirus Particle”, “Alphavirus replicon particle” and“Replicon particle” refers to a virion unit containing an alphavirus RNAvector replicon. Generally, the recombinant alphavirus particlecomprises one or more alphavirus structural proteins, a lipid envelopeand an RNA vector replicon. Preferably, the recombinant alphavirusparticle contains a nucleocapsid structure that is contained within ahost cell-derived lipid bilayer, such as a plasma membrane, in which oneor more alphaviral envelope glycoproteins are embedded. The particle mayalso contain other components (e.g., targeting elements such as biotin,other viral structural proteins, hybrid envelopes, or other receptorbinding ligands) which direct the tropism of the particle from which thealphavirus was derived.

“Alphavirus packaging cell line” refers to a cell which contains one ormore alphavirus structural protein expression cassettes and whichproduces recombinant alphavirus particles after introduction of analphavirus RNA vector replicon, eukaryotic layered vector initiationsystem, or recombinant alphavirus particle. The parental cell may be ofmammalian or non-mammalian origin. Within preferred embodiments, thepackaging cell line is stably transformed with the structural proteinexpression cassette(s).

“Eukaryotic Layered Vector Initiation System” refers to an assembly thatis capable of directing the expression of a sequence or gene ofinterest. The eukaryotic layered vector initiation system should containa 5′ promoter which is capable of initiating in vivo (i.e. within aeukaryotic cell) the synthesis of RNA from cDNA, and a nucleic acidvector sequence (e.g., viral vector) which is capable of directing itsown replication in a eukaryotic cell and also expressing a heterologoussequence. Preferably, the nucleic acid vector sequence is analphavirus-derived sequence and is comprised of a 5′ sequence which iscapable of initiating transcription of an alphavirus RNA (also referredto as 5′ viral sequences required in cis for replication or 5′ CSE), aswell as sequences which, when expressed, code for biologically activealphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and analphavirus RNA polymerase recognition sequence (also referred to as 3′viral sequences required in cis for replication or 3′ CSE). In addition,the vector sequence may include an alphaviral subgenomic “junctionregion” promoter which may, in certain embodiments, be modified in orderto prevent, increase, or reduce viral transcription of the subgenomicfragment, as well as a polyadenylation sequence. The eukaryotic layeredvector initiation system may also contain splice recognition sequences,a catalytic ribozyme processing sequence, a nuclear export signal,heterologous gene, and a transcription termination sequence. In certainembodiments, in vivo synthesis of the vector nucleic acid sequence fromcDNA may be regulated by the use of an inducible promoter or subgenomicexpression may be inducible through the use of translational regulatorsor modified nonstructural proteins.

“External Component Bioreactor” refers to an integrated modularbioreactor system for the mass culture, growth, and process control ofsubstrate attached cells. The External Component Bioreactor should havea vessel or chamber with tissue culture treated growth surface forattachment and propagation of cells (e.g., alphavirus packaging cells).Unlike traditional “stir-tank” bioreactors, which may have an internalmechanical agitation (e.g., impeller) and/or sparging system tocirculate culture media and facilitate gas exchange, the ExternalComponent Bioreactor should have external components or modules that areconnected (i.e., via tubing), to achieve similar functions. In certainembodiments, the external components may include pumps, reservoirs,oxygenators, culture modules, and other non-standard parts.

“Tentacle ion exchange resin” refers to a resin, gel or matrix withfunctional charge groups and wherein the functional charge groups arecarried by long polymer chains (“tentacles”), rather than being locatedon the surface of the resin, gel or matrix. In certain embodiments, thetentacle ion exchange resin is a cationic resin, gel or matrix that maybe used to bind and fractionate biological substances on the basis ofcharge characteristics. A representative example of a tentacle cationicexchange resin is Fractogel® EMD SO₃ ⁻ (M) (s-Fractogel®). In otherembodiments, the tentacle ion exchange resin is an anionic resin, gel ormatrix that may be used to bind and fractionate biological substances onthe basis of charge characteristics. A representative example of atentacle anionic exchange resin is Fractogel® EMD DEAE (M).

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription, which provides illumination of the practice of theinvention.

DETAILED DESCRIPTION OP THE INVENTION

As noted above, the present invention provides methods of purificationfor viruses and virus-derived vectors, including those related toalphaviruses, from biological and chemical preparations. In particular,this invention relates to methods of purification for such viruses andvectors from preparations by subjecting the preparation tochromatographic purification methods, such as for example, using atentacle cationic exchange resin with or without a secondchromatographic purification step. Viruses and vectors purifiedaccording to this invention have use as effective vaccines andtherapeutics.

Alphavirus Vector Replicons And Replicon Particles

As noted above, the present invention provides replicons and repliconparticles derived from a wide variety of alphaviruses. Such repliconsand particles, including sequences encoding alphaviruses suitable foruse in preparing the above-described materials, have been described indetail elsewhere (see, for example, U.S. Pat. Nos. 5,789,245, 5,842,723,and 6,015,694; PCT Nos. WO 97/38087, WO 99/18226, WO 00/61772, and WO00/39318), which are hereby incorporated by reference in their entirety.

Heterologous Sequences

A wide variety of nucleotide sequences may be carried and expressed bythe alphavirus replicon particles of the present invention, including,for example, sequences which encode lymphokines, cytokines, orchemokines (e.g., IL-2, IL-12, GM-CSF, SLC), prodrug converting enzymes(e.g., HSV-TK, VZV-TK), antigens which stimulate an immune response(e.g., HIV, HCV, tumor antigens), therapeutic molecules such as growthor regulatory factors (e.g., VEGF, FGF, PDGF, BMP), proteins whichassist or inhibit an immune response, as well as ribozymes and antisensesequences. The above nucleotide sequences include those referencedpreviously (e.g., U.S. Pat. No. 6,015,686, WO 97/38087 and WO 99/18226,WO 00/61772, and WO 00/39318), and may be obtained from repositories,readily cloned from cellular or other RNA using published sequences, orsynthesized, for example, on an Applied Biosystems Inc. DNA synthesizer(e.g., APB DNA synthesizer model 392 (Foster City, Calif.)).

Alphavirus Replicon Particle Production

Alphavirus replicon particles according to the present invention may beproduced using a variety of published methods. Such methods include, forexample, transient packaging approaches, such as the co-transfection ofin vitro transcribed replicon and defective helper RNA(s) (Liljestrom,Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., J. Virol.67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997; Pushkoet al., Virology 239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and5,842,723) or plasmid DNA-based replicon and defective helper constructs(Dubensky et al., J. Virol. 70:508-519, 1996), as well as introductionof alphavirus replicons into stable packaging cell lines (PCL) (Polo etal., PNAS 96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245, 5,842,723,6,015,694; WO 97/38087, WO 99/18226, WO 00/61772, and WO 00/39318).

It should be noted that the selected method for production of repliconparticles should preferably minimize or eliminate the possibility ofgenerating contaminating replication-competent virus (RCV). One suchstrategy to address this issue of RCV is the use of defective helpers orPCL that contain “split” structural protein expression cassettes (seeU.S. Pat. No. 5,789,245). In this context, the alphavirus structuralprotein genes are segregated into separate expression constructs (e.g.,capsid separate from glycoproteins) such that multiple recombinationevents are required to regenerate a complete complement of structuralproteins, which is extremely unlikely.

In preferred embodiments, stable alphavirus packaging cell lines areutilized for replicon particle production (FIG. 1). The PCL may betransfected with in vitro transcribed replicon RNA, transfected withplasmid DNA-based replicon (e.g., ELVIS vector), or infected with a seedstock of replicon particles, and then incubated under conditions and fora time sufficient to produce high titer packaged replicon particles inthe culture supernatant. In particularly preferred embodiments, PCL areutilized in a two-step process, wherein as a first step, a seed stock ofreplicon particles is produced by transfecting the PCL with a plasmidDNA-based replicon. A much larger stock of replicon particles is thenproduced in the second step, by infecting a fresh culture of the PCLwith the seed stock. This infection may be performed using variousmultiplicities of infection (MOI), including a MOI=0.01, 0.05, 0.1, 0.5,1.0, 3, 5, or 10. Preferably infection is performed at a low MOI (e.g.,less than 1). Replicon particles at titers >10⁸ infectious units (IU)/mlcan be harvested over time from PCL infected with the seed stock. Inaddition, the replicon particles can subsequently be passaged in yetlarger cultures of naive PCL by repeated low multiplicity infection,resulting in commercial scale preparations with the same high titer.Importantly, by using PCL of the “split” structural gene configuration,these replicon particle stocks are free from detectable contaminatingRCV.

As described above, large-scale production of alphavirus repliconparticles may be performed using a bioreactor. Preferably, thebioreactor is an External Component Bioreactor, which is an integratedmodular bioreactor system for the mass culture, growth, and processcontrol of substrate attached cells. The attachment and propagation ofcells (e.g., alphavirus packaging cells) occurs in a vessel or chamberwith tissue culture treated surfaces, and the cells are with fresh mediafor increased cell productivity. Monitoring and adjustments areperformed for such parameters as gases, temperature, pH, glucose, etc.,and crude vector is harvested using a perfusion pump. Typically, theindividual components of an External Bioreactor separate externalmodules that are connected (i.e., via tubing). The external componentscan be pumps, reservoirs, oxygenators, culture modules, and othernon-standard parts. A representative example of an External ComponentBioreactor is the CellCube™ system (Corning, Inc).

In addition to using the External Component Bioreactor described herein,a more traditional Stir Tank Bioreactor may also be used, in certaininstances, for alphavirus replicon particle production. In a Stir TankBioreactor, the alphavirus packaging cells may be unattached to anymatrix (i.e., floating in suspension) or attached to a matrix (e.g.,poly disks, micro- or macro carriers, beads). Alternatively, a HollowFiber Culture System may be used.

Purification Using Ion Exchange Resins

Following harvest, crude culture supernatants containing the alphavirusreplicon particles may be clarified by passing the harvest through afilter (e.g., 0.2 uM, 0.45 uM, 0.65 uM, 0.8 uM pore size). Optionally,the crude supernatants may be subjected to low speed centrifugationprior to filtration to remove large cell debris. Within one embodiment,an endonuclease (e.g., Benzonase, Sigma #E8263) is added to thepreparation of alphavirus replicon particles before or after achromatographic purification step to digest exogenous nucleic acid.Further, the preparation may be concentrated prior to purification usingone of any widely known methods (e.g., tangential flow filtration).

Crude or clarified alphavirus replicon particles may be concentrated andpurified by chromatographic techniques (e.g., ion exchangechromatography, size exclusion chromatography, hydrophobic interactionchromatography, affinity chromatography). Two or more such purificationmethods may be performed sequentially. In preferred embodiments, atleast one step of ion exchange chromatography is performed and utilizesa tentacle ion exchange resin. Briefly, clarified alphavirus repliconparticle filtrates may be loaded onto a column containing a charged ionexchange matrix or resin (e.g., cation or anion exchange). The matrix orresin may consist of a variety of substances, including but not limitedto cross linked agarose, cross linked polystyrene, cross linked styrene,hydrophilic polyether resin, acrylic resin, and methacrylate basedresin. The ion exchanger component may comprise, but is not limited to,a cationic exchanger selected from the list consisting of sulphopropylcation exchanger, a carboxymethyl cation exchanger, a sulfonic acidexchanger, a methyl sulfonate cation exchanger, and an SO3-exchanger. Inother embodiments, the ion exchanger component may comprise, but is notlimited to, an anionic exchanger selected from the list consisting ofDEAE, TMAE, and DMAE. Most preferably, ion exchange chromatography isperformed using a tentacle cationic exchanger, wherein the ion exchangeresin is a methacrylate-based resin with an SO3-cation exchanger (e.g.,Fractogel® EDM SO3-).

The replicon particles may be bound to the ion exchange resin followedby one or more washes with buffer containing a salt (e.g., 250 mM orless NaCl). Replicon particles then may be eluted from the column inpurified form using a buffer with increased salt concentration. Inpreferred embodiments, the salt concentration is a least 300 mM, 350 mM,400 mM, 450 mM or 500 mM. Elution may be monitored preferably by aspectrophotometer at 280 nm, but also by replicon titer assay, transferof expression (TOE) assay, or protein gel analysis with subsequentCoomassie staining or Western blotting.

The higher salt elution buffer subsequently may be exchanged for a moredesirable buffer, for example, by dilution in the appropriate aqueoussolution or by passing the particle-containing eluate over a molecularexclusion column. Additionally, the use of a molecular size exclusioncolumn may also provide, in certain instances, further purification. Forexample, in one embodiment Sephacryl S-500 or S-400 (Pharmacia)chromatography may be used as both a buffer exchange as well as tofurther purify the fractions containing the replicon particles elutedfrom an ion exchange column. Using this particular resin, the repliconparticles generally are eluted in the late void volume and showimprovement in the level of purity as some of the contaminants aresmaller in molecular weight and are retained on the column longer.However, alternative resins of different compositions as well as sizeexclusion could also be used that might yield similar or improvedresults. In these strategies, larger-sized resins such as SephacrylS-1000 could be incorporated that would allow the replicon particles toenter into the matrix and thus be retained longer, allowingfractionation.

Methods of Determining Replicon Particle Titer

Two methods of titering alphavirus replicon particles are widelyaccepted in the viral vector field. The first method of titering is asimple transfer of expression assay, wherein a culture of naïve cells isinfected with various dilutions (e.g., serial dilutions) of the unknownreplicon particle preparation and individual cells expressing theencoded gene of interest are quantitated to arrive at original titer.Identification of cells expressing the encoded gene of interest may beperformed according to the specific protein being expressed (e.g.,fluorescence for GFP reporter, chemical staining for β-gal,immunocytochemistry for proteins with available antibody).Alternatively, an alphavirus reporter cell line (e.g., Olivo et al.,Virology 198:381-384, 1984) may be used in conjunction with repliconparticles expressing a reporter gene, which serve as a known titerstandard curve. Values for the unknown, obtained after infection of thereporter cell line with various dilutions, can be extrapolated tocalculate titer.

The present invention discloses additional methods of quantitatingreplicon particles in a preparation, and these methods are not limitedon a preparation by preparation basis, such as from one gene of interestto another. The first method is based upon nucleic acid detection andamplification of the nucleic acid product or a signal that is specificto the assay. Such methods can include, for example, PCR, TMA and BDNA(branched DNA) assays. These nucleic acid based assays provide extremelysensitive levels of detection. More specifically, in the case of a bDNAbased assay, a single-stranded DNA probe that is specific and unique toa region of alphavirus genomic and replicon RNA was designed. This probeis bound to the bDNA plate. Target cells that have been infected withserial dilutions of replicon particle preparations are lysed anddirectly transferred to the bDNA plate. After overnight incubation, thealphavirus genomic RNA hybridizes to the homologous single-stranded DNAprobe. The plate is then washed to clear non-specific material, andsequentially incubated with a series of hybridization amplifiers. Thesignal generated is luminescence-based and can be analyzed in aspectrophotometer. A standard curve can be generated using reporterreplicon particle preparations of known titer, for example vectorencoding β-galactosidase or green fluorescent protein. The titer of theunknown sample is determined by extrapolation.

The second method of quantitation is by complementation of the repliconvector so as to allow detection by plaque assay in cultured cells.Replication defective viral vectors, such as alphavirus replicons, whichare deleted of one or more genes encoding structural proteins necessaryfor packaging are considered “suicide vectors” and cannot spread fromcell to cell. As such, traditional plaque assay methods of quantitationare impossible. The present invention provides a method of performingplaque assay by using packaging cells which express the necessarystructural proteins required for production of progeny particles. Thepackaging cells used for such an assay may contain one or morestructural protein expression cassettes. In the case of alphavirusreplicon particles, packaging cells are infected with serial dilutionsof replicon particle preparations, overlayed and plaques enumerated.

Pharmaceutical Compositions

As noted above, the present invention also provides pharmaceuticalcompositions comprising purified alphavirus replicon particles incombination with a pharmaceutically acceptable carrier, diluent, orrecipient. As used herein, purified shall mean an alphavirus repliconparticle preparation free from detectable non-alphavirus proteins.Detection of non-alphavirus proteins is determined by gelelectrophoresis using a sample size of between approximately 10⁸ to 10⁹replicon particles. Gel electrophoreiss methods including, but notlimited to polyacrylamide gel electrophoresis (PAGE), discelectrophoresis and SDS-PAGE, followed by standard Coomassie staining.More specifically, “purified” shall mean alphavirus particlepreparations subjected to multi-step chromatography purificationprocedures as disclosed herein. Within certain preferred embodiments, asufficient amount of formulation buffer is added to the purifiedreplicon particles to form an aqueous suspension. In preferredembodiments, the formulation buffer comprises a saccharide and abuffering component in water, and may also contain one or more aminoacids or a high molecular weight structural additive. The formulationbuffer is added in sufficient amount to reach a desired finalconcentration of the constituents and to minimally dilute the repliconparticles. The aqueous suspension may then be stored, preferably at −70°C., or immediately dried.

The aqueous suspension can be dried by lyophilization or evaporation atambient temperature. Briefly, lyophilization involves the steps ofcooling the aqueous suspension below the gas transition temperature orbelow the eutectic point temperature of the aqueous suspension, andremoving water from the cooled suspension by sublimation to form alyophilized replicon particle. Within one embodiment, aliquots of theformulated recombinant virus are placed into an Edwards RefrigeratedChamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo12K). A multistep freeze drying procedure as described by Phillips etal. (Cryobiology 18:414, 1981) is used to lyophilize the formulatedreplicon particles, preferably from a temperature of −40° C. to −45° C.The resulting composition contains less than 10% water by weight of thelyophilized replicon particles. Once lyophilized, the replicon particlesare stable and may be stored at −20° C. to 25° C., as discussed in moredetail below. In the evaporative method, water is removed from theaqueous suspension at ambient temperature by evaporation. Within oneembodiment, water is removed by a spray-drying process, wherein theaqueous suspension is delivered into a flow of preheated gas, usuallywhich results in the water rapidly evaporating from droplets of thesuspension. Once dehydrated, the recombinant virus is stable and may bestored at −20° C. to 25° C.

The aqueous solutions used for formulation preferably comprise asaccharide, a buffering component, and water. The solution may alsoinclude one or more amino acids and a high molecular weight structuraladditive. This combination of components acts to preserve the activityof the replicon particles upon freezing and also lyophilization ordrying through evaporation. Although a preferred saccharide is lactose,other saccharides may be used, such as sucrose, mannitol, glucose,trehalose, inositol, fructose, maltose or galactose. A particularlypreferred concentration of lactose is 3%-4% by weight.

The high molecular weight structural additive aids in preventingparticle aggregation during freezing and provides structural support inthe lyophilized or dried state. Within the context of the presentinvention, structural additives are considered to be of “high molecularweight” if they are greater than 5000 M.W. A preferred high molecularweight structural additive is human serum albumin. However, othersubstances may also be used, such as hydroxyethyl-cellulose,hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone. Aparticularly preferred concentration of human serum albumin is 0.1% byweight.

The buffering component acts to buffer the solution by maintaining arelatively constant pH. A variety of buffers may be used, depending onthe pH range desired, preferably between 7.0 and 7.8. Suitable buffersinclude phosphate buffer and citrate buffer. In addition, it ispreferable that the aqueous solution contains a neutral salt that isused to adjust the final formulated replicon particles to an appropriateiso-osmotic salt concentration. Suitable neutral salts include sodiumchloride, potassium chloride or magnesium chloride. A preferred salt issodium chloride. The lyophilized or dehydrated replicon particles of thepresent invention may be reconstituted using a variety of substances,but are preferably reconstituted using water. In certain instances,dilute salt solutions that bring the final formulation to isotonicitymay also be used.

Methods For Delivery of Replicon Particles

As noted above, the present invention also provides methods fordelivering a selected heterologous sequence to a warm-blooded mammal(e.g., a mammal such as a human or other warm-blooded animal such as ahorse, cow, pig, sheep, dog, cat, rat or mouse) for use as a vaccine ortherapeutic, comprising the step of administering to the mammal repliconparticles purified and/or characterized as described herein. Deliverymay be by a variety of routes (e.g., intravenously, intramuscularly,intradermally, intraperitoneally, subcutaneously, orally, intraocularly,intranasally, rectally, intratumorally). In addition, the repliconparticles may either be administered directly (i.e., in vivo), or tocells which have been removed (ex vivo), and subsequently returned tothe warm-blooded mammal.

The following examples are included to more fully illustrate the presentinvention. Additionally, these examples provide preferred embodiments ofthe invention and are not meant to limit the scope thereof.

EXAMPLES Example 1 Production of Alphavirus Replicon Particles Using APackaging Cell Line

To demonstrate scalability of replicon particle production in adherentcultures of an alphavirus packaging cell line, experiments wereperformed in either a 10-tray Nunc Cell Factory or a Corning Cell Cube.For example, 2.5×10⁸ cells of an alphavirus packaging cell line, PCL#15.25, which expresses human dendritic cell tropic Sindbis structuralproteins (Gardner et al., J. Virol., 74:11849-11857, 2000) weresuspended in 100 ml of Dulbecco's Modified Eagle's Medium (DMEM)supplemented with penicillin, streptomycin, L-glutamine, and 1% fetalcalf serum (FCS). To this suspension, 1.26×10⁸ SIN replicon particlesencoding a GFP reporter (Gardner et al., 2000, ibid) were added at amultiplicity of infection (MOI) of approximately 0.57 particles percell. The suspension was incubated at 37° C. and gently mixed every 15minutes for approximately 1.5 hours. The suspension was then added to 1liter of pre-warmed (37° C.) DMEM with 5% FCS, transferred to a 10-trayNunc Cell Factory, and placed in an incubator set at 34° C., 5% CO₂.Complete media exchanges were made at 22 hr, 30 hr, 44 hr, 52 hr, 70 hr,78 hr, and 90 hr post-infection and replicon particle titers weredetermined for each harvest (FIG. 2). Culture fluids collected for thehighest titer harvests (harvests 1-5) were pooled, transferred tocentrifuge bottles, and cell debris was pelleted by centrifugation at2,500 RPM in a Sorvall RT6000 centrifuge, at 4° C. for 15 minutes. Thesupernatant then was passed through a 0.2 um cellulose acetatefiltration unit and used for chromatographic purification as describedin example 2 below. Similar production runs have been performed usingmore than one Cell Factory, in order to increase the total harvest ofalphavirus replicon particles proportionally.

Large-scale manufacture of alphavirus replicon particles also may beaccomplished, for example, using the CellCube™ bioreactor system (FIG.3). The CellCube™ system is an integrated modular bioreactor withmulti-layer (100-stack) growth chambers of 85,000 cm² surface area.Controlling the mixtures of oxygen, CO₂ and air allows precise controlof pH and DO₂ parameters. Together with glucose monitoring andadjustment, this level of culture control provides an increased capacityfor replicon particle production.

PCL-based production runs of alphavirus replicon particles using theCellCube™ system requires the input of an initial seed stock of repliconparticles to be amplified. To demonstrate feasibility of this approach,CellCube™ production was performed by expanding PCL successively in T225cm² cell culture flasks, and increasing to a surface area of four10-layer Cell Factories prior to suspension infection with the particleseed stock. The PCL were trypsinized using a minimal amount of trypsin,diluted with growth media and then centrifuged briefly. Resuspendedcells were counted, split into equal halves, and infected with the seedstock of replicon particles at low MOI infection. Infection was allowedto proceed in suspension with gentle agitation for 30 minutes. After 30minutes, one vessel was placed on ice and the other was transferred intothe inoculation carboy containing 7 L of 5% FBS DMEM inoculation media.The 7 L of infected cell suspension was transferred into the CellCubeand the culture module was rotated 90° to enable the cells to attach.After 60 minutes, the suspension was drained back into the inoculationcarboy and the second vial of infected PCL (from ice) was added. Thissuspension, like the first, was transferred into the culture module,which was then rotated 180° to enable these cells to attach to theopposite side of the culture support plates. After 3 hours, the systemwas rotated back to the horizontal position and back-filled with 5%DMEM, 20 mM HEPES, after which, circulation was initiated, and gasseswere adjusted to maintain pH and DO2 levels. Daily sampling allowedtesting and profiling of metabolic indicators including glucose levels.The perfusion system was adjusted based on glucose consumption andprevious data to ensure maximum yield of vector. Automated andcontinuous harvest into 4° C. vessels was used to minimizetemperature-induced degradation of replicon particles and allow maximumyield and highest ratio of viable replicon particles.

The initial CellCube T production runs illustrated for this example wereperformed using early generation replicon and packaging cell lines(prior to the derivation of PCL #15-25 above). These earlier versions ofreagents are known to yield alphavirus replicon particles at lowertiters (Polo et al., 1999, PNAS 96:4598-4603) than the reagents nowavailable, however such techniques are identical to those that would beused for any subsequently derived vector replicon and packaging cellline. The data obtained using these reagents indicate that large-scale(90-100 liter) production lots can be generated in a 100-stack CellCube™module, with the same titer efficiency as small, research-scalepackaging experiments (FIG. 4). Expanded CellCube™ systems that employfour 100-stack modules, thus, have the potential to readily produce 400L+ of replicon particle production material per run.

To generate seed stocks of alphavirus replicon particles without a priorstep of in vitro transcription, the packaging cell lines may betransfected with a plasmid DNA-based replicon (Eukaryotic Layered VectorInitiation System) encoding the heterologous gene of interest.Large-scale transfections are carried out in Nunc 10 layer CellFactories, using the calcium phosphate method according to the followingparameters. Packaging cells are plated in the Cell Factory one day priorto transfection at a density of 8×10⁴ cells/cm². The DNA:calciumphosphate mixture is prepared in a volume of 200 ml, diluted with 1liter of media and added to the packaging cell line in the Cell Factory.The media is exchanged after 6-8 hr and replicon particle seed stockmaterial is harvested in multiple batches, over a period of 2-3 days.Harvests are pooled, purified, and aliquoted for long-term storage at−80° C. Alphavirus replicon particle seed stock material then may beused for subsequent large-scale amplifications in naïve PCL (e.g., inCellCube bioreactor). Alternative methods of transfecting the plasmidDNA-based replicon also may be substituted readily by one of skill inthe art, including but not limited to lipid-mediated transfection andelectroporation.

Example 2 Purification of Replicon Particles Using s-Fractogel® CationicExchange Resin

To compare the efficiency of replicon particle purification using atentacle cationic exchange resin, Fractogel® EMD SO₃ ⁻ (M)(s-Fractogel®, EM Industries), with the ion exchange resin Matrix®Cellufine™ Sulfate (Amicon), columns of the same size were equilibratedwith 10 mM sodium phoshate, 125 mM sodium chloride, pH 7.0. Clarifiedculture supernatants. (˜260 ml) containing SIN-GFP replicon particlesgenerated as described (Polo et al., PNAS 96:4598-4603, 1999) werepassed through s-Fractogele and Cellufine™ Sulfate columns at flow ratesof 115 and 75 cm/hour respectively. The columns were washed withapproximately 20-40 column volumes of buffer containing 10 mM sodiumphoshate, 250 mM sodium chloride, pH 7.0, and bound SIN-GFP repliconparticles were eluted in a 20 ml, 0.5M-2.0M NaCl linear gradientcollected in 1 ml fractions. A final 3 M NaCl rise was then used toremove any remaining replicon particles.

For analysis, consecutive fractions were pooled in pairs starting withfractions 2 and 3, continuing with 4 and 5, etc. Replicon particletiters (total IU) were determined for the recovered fractions, as wellas the starting material, load, and wash (FIG. 5). Based on the titerassay results, the 260 ml of clarified supernatant starting materialcontained approximately 2.4×10¹⁰ IU total. Recovery in the main elutionpeaks from the s-Fractogel® column was 1.3×10¹⁰ IU total, orapproximately 55% of the load, with almost all (99%) concentrated inpooled fractions 2 and 3. Subsequent purification runs using s-Fractogelindicated an average recovery of 80-90%. In contrast, the total recoveryfrom the Cellufine™ Sulfate column was consistently lower, and for thisexperiment was approximately 3.0×10⁸ IU total (or <2%) in the two mainfractions, thus resulting in a considerably more dilute product.

Samples also were analyzed for purity by subjecting the collectedfractions to polyacrylamide gel electrophoresis (Coomassie staining,FIG. 6) and Western blotting (not shown). The results of 10-20% SDS PAGECoomassie-stained gels indicated an improvement in the purity of thes-Fractogel® peak as compared to the Cellufine™ Sulfate peak (see SINparticle capsid and glycoprotein bands in sample 2). Interestingly, themain peak of recovered particles, found in pooled fractions 2 and 3 fromeach column, eluted just prior to a considerable peak of contaminants inpooled fraction 4 and 5 from each column. If fraction 4/5 material wasexcluded from the Cellufine™ Sulfate pooled products due to theincreased amount of impurity, it would reduce the effective recoveryeven further.

In addition to the improved efficiency of purification, the cost basisof the s-Fractogel® is considerably lower than for the Cellufine™Sulfate. Cost analysis for the resin component only indicates anapproximately 3-fold cost decrease with s-Fractogel®, assuming thatequal amount of resins could be used. However, the data suggest thatthere may have been an overloading on the Cellufine™ Sulfate column andthat additional resin may be required for equivalent binding capacity.Finally, the reduced flow rate of the Cellufine™ Sulfate column wouldtranslate to 50% increase in column run time and thus, another increasedcost per run. Taken together, the s-Fractogel® purification method ofthe present invention provides superior overall utility for large-scalecommercial manufacture of alphavirus replicon particles.

In additional experiments, increased volumes of alphavirus repliconparticles (e.g., those generated using at least a Cell Factory) alsowere purified using the s-Fractogel® methodology. For example, a total25 ml of s-Fractogel® was packed in a Pharmacia AK-26 column andequilibrated with 20 column volumes of buffer (10 mM sodium phosphate,pH 7.0 and 125 mM NaCl) at a linear flow rate of 115 cm/hour. Afterequilibration, approximately 5.5 liters of culture supernatantcontaining alphavirus replicon (see Example 1) was passed over thecolumn. The column was washed with approximately 300 ml of wash buffer(10 mM sodium phosphate, pH7.0, 250 mM NaCl), and the particles wereeluted in 12 ml fractions by buffer containing 10 mM sodium phosphate,pH7.0, 400 mM NaCl.

Determination of recovery and identification of peak fractionscontaining the alphavirus replicon particles was performed by titerassay in which aliquots from the starting material, the flow through,the wash, and the eluted fractions were serially diluted and used toinfect BHK-21 cells. The results from this purification procedure (FIG.7) indicate that the 5.5 liters of harvested supernatant containedreplicon particles with a titer of approximately 1.2×10⁸ IU/ml and thatonly a negligible amount of particles was found in either the flowthrough or the wash. The highest concentration of eluted, purifiedparticles was found in the 2^(nd) and 3^(rd) fractions eluted from thecolumn at a concentration of 1.3×10¹¹ and 1.4×10¹⁰ IU/ml.

Example 3 Stimulation of the Immune Response Using Alphavirus RepliconParticles

To demonstrate the potent stimulation of antigen specific immuneresponses using purified alphavirus replicon particles, the sequenceencoding HIV-1 p55gag was inserted into SIN-based replicons. The HIV gagcoding sequence was selected from the HIV-1SF2 strain (Sanchez-Pescador,R., et al., Science 227 (4686):484-492, 1985; Luciw, P. A., et al., U.S.Pat. No. 5,156,949, herein incorporated by reference; Luciw, P. A., etal., U.S. Pat. No. 5,688,688). These sequences have been used directlyor first manipulated to maximize expression of their gene products. Formaximization of expression, the HIV-1 codon usage pattern was modifiedso that the resulting nucleic acid coding sequence was comparable tocodon usage found in highly expressed human genes. The HIV codon usagereflects a high content of the nucleotides A or T as third base of thecodon-triplet. The effect of the HIV-1 codon usage is a high AT contentin the DNA sequence that could result in a decreased translation abilityand instability of the mRNA. In comparison, highly expressed humancodons prefer the nucleotides G or C as the third base. The gag codingsequence therefore was modified to be comparable to codon usage found inhighly expressed human genes.

The DNA fragment for gag first was cloned into the eukaryotic expressionvector pCMVKm2, derived from pCMV6a (Chapman et al., Nuc. Acids Res.19:3979-3986, 1991), to generate the construct pCMVKm2.GagMod.SF2. Thisplasmid was deposited Jan. 18, 1999, with the Chiron Corporation MasterCulture Collection, Emeryville, Calif., 94662-8097, and with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. The HIV gag gene then was subcloned into a SIN repliconvector (SINCR, Gardner et al., ibid) for the generation of alphavirusreplicon particles by digestion with EcoRI, blunt-ending with Klenow anddNTPs, purification with GeneCleahII, and digestion with SalI. The HIVgag-coding fragment then was ligated into the SINCR vector that had beendigested with NotI, blunt-ended, and digested with XhoI. The resultingvector was designated SINCR-gag.

To compare the effective purification as well as demonstrate themaintenance of immunogenicity of column-purified replicon particles, aproduction run of 4×10-tray Nunc Cell Factories was undertaken.Approximately 2×10⁹ cells of alphavirus packaging cell line, PCL #15.25,were suspended in 400 ml of Dulbecco's Modified Eagle's Medium (DMEM)supplemented with penicillin, streptomycin, L-glutamine, and 1% fetalcalf serum (FCS). To this suspension, 1×10¹⁰ SIN replicon particlesencoding HIV p55 Gag were added at a multiplicity of infection (MOI) ofapproximately 5. The suspension was incubated at 37° C. and gently mixedevery 15 minutes for approximately 1 hour. The suspension was thendivided into 4×100 ml aliquots and each 100 ml aliquot was added to 1liter of pre-warmed (37° C.) DMEM with 5% FCS, transferred to the10-tray Nunc Cell Factory, and placed in an incubator set at 34° C., 5%CO₂. Complete media exchanges were made at 20 hr, 28 hr, and 40 hrpost-infection. Culture fluids collected from at each harvest wastransferred to centrifuge bottles, and cell debris was pelleted bycentrifugation at 2,500 RPM in a Sorvall RT6000 centrifuge, at 4° C. for15 minutes and the resulting supernatant was passed through a 0.2 μmcellulose acetate filtration unit. Approximately 8 L of supernatant wasloaded onto a 2.6 cm diameter column containing 30 ml of s-Fractogelresin equilibrated with 10 mM sodium phoshate, 125 mM sodium chloride,pH 7.0. The flow rate of 58 cm/hour was used for the first 5 liters and115 cm/hour for the last 3 L. The column was rinsed with 10 mM sodiumphosphate, 125 mM sodium chloride, pH 7.0 and was followed by two washsteps containing 10 mM sodium phosphate, 250 mM sodium chloride, pH 7.0then 10 mM sodium phosphate, 300 mM sodium chloride. The particles wereeluted with 10 mM sodium phosphate, 400 mM sodium chloride, pH 7.0. Thetwo peak s-Fractogel fractions (#2 and #3) were pooled and 10 ml of thepool was loaded onto a Sephacryl S-400 HR (Pharmacia) (diameter=2.6 cm,column volume=490 ml) equilibrated with buffer containing 40 mg/mllactose in PBS. The flow rate was 3.3 ml/minute and each fractioncontained 12 ml. Samples from the s-Fractogel and the S-400 wereanalyzed for titer recovery as well as purity by as determined bypolyacrylamide gel electrophoresis Coomassie and silver staining. Basedon the titer assay, the 4-cell factory run generated approximately1×10¹² IU total particles. Approximately 8×10¹¹ IU were loaded onto thes-Fractogel column and approximately 6×10¹¹ IU were eluted in the mainpeak yielding a 75% recovery. From the s-Fractogel peak, 3×10¹¹ IU wereloaded onto the S-400 column, with approximately 1.5×10¹¹ IU eluted inthe main peak resulting in a 50% recovery. The relative purity of thes-Fractogel and the S-400 samples are shown in FIG. 8.

In order to determine if the purified SIN replicon particles encodingHIV-p55 maintained immunogenicity, a study was designed to compare thepurified particles with an essentially unpurified, but concentrated(polyethelyene glycol precipitation) preparation of particles using anGag-specific IFN-γ ELISPOT. In the study, mice (5 mice per group) wereimmunized with SIN-gag replicon particle preparations (10⁶ IU/animal)that were PEG precipitated, purified with a single step of cationicexchange chromatography, or a two step process of cationic exchangechromatography followed by size exclusion chromatography. Animalsreceived immunizations at days 0 and 21 with sample collection at days29 and 30.

To measure the number of Gag-specific IFN-γ secreting cells, an ELISPOTassay was performed. Single-cell suspensions from pooled cervical lymphnodes and spleens from the mice in each group were added ontonitrocellulose or pvdf plates (Millipore) pre-coated with monoclonal ratanti-mouse anti-IFN-γ antibody (Pharmingen) and blocked with completeRPMI medium at pH 7.2, containing 10% fetal calf serum, 5 mM Hepes, andantibiotics. Following overnight incubation of cells in the presence ofgag-derived p7g peptide, or anti-CD3 (Pharmingen) and anti-CD28(Pharmingen) as positive control for polyclonal T cell activation, ormedia only as negative control, the plates were washed and biotinylatedanti-IFNγ (Pharmingen) was added in PBS/0.1% BSA/0.02% Tween andincubated at R/T for 2 hours. The plates were washed with P/T andincubated for 1 hr at 37° C. with Avidin-peroxidase (Pharmingen) at1:1000 dilution. The plates were washed with P/T and the spots werevisualized by adding DAB in Tris-HCl (pH 7.5) buffer for 30 minutes. Theplates were washed with de-ionized H2O and air-dried. Background spotsfrom negative control (media only) wells were subtracted from wellsactivated with gag-p7g peptide. The number of spots in positive controlwells (polyclonally activated with anti-CD3 and anti-CD28) was 5-10 foldhigher than the number of spots in wells activated with gag-p7g peptide.The spots were counted with an in-house developed automated ELISPOTreader using software from Alpha Innotech Corporation (San Leandro,Calif.).

The results shown in FIG. 9 are representative of two independentexperiments from two pools of each group expressed as the number ofgag-p7g peptide-specific IFN-γ secreting cells per 10⁷ mononuclearcells. The results indicate no loss of immunogenicity from either methodof purification.

Similarly, the stimulation of an antitumor response was demonstrated inthe widely accepted CT26 colon carcinoma system by administering SINderived alphavirus replicon particles expressing the cytokine IL-2. TheIL-2 gene was inserted into the SIN replicon vector following PCRamplification and replicon particles were produced using methodsdescribed above. On four successive days following tumor inoculation,mice were injected intratumorally with 10⁸ SIN-IL2 replicon particles.Additional animals received as controls the diluent only, recombinantIL2 protein which has an established clinical efficacy in humans, orSIN-GFP particles. Animals were monitored for increased tumor volume andgroup means for each treatment group arm were compared. When the groupmean for a given arm (e.g., diluent control) reached 2000 mm³, theanimals were euthanized compared. As seen in FIG. 10, SIN-IL2 treatedanimals showed a significant anti-tumor response that was at leastcomparable to the recombinant IL2 protein.

Example 4 Characterization of Alphavirus Replicon Particles

To quantitate the number of replicon particles in a preparation, twonovel methods are disclosed herein. In the first instance, stablealphavirus packaging cell lines (see for example U.S. Pat. No.5,789,245, U.S. Pat. No. 5,843,723, and WO 99/18226) are provided. Thepackaging cell lines express each of the alphavirus structural proteins(e.g., capsid, glycoproteins) necessary for production of alphavirusparticles, which are not encoded by an alphavirus replicon vectoritself. Packaging cell line #15-25 (see above) cells were plated in6-well dishes to achieve approximately 80-90% confluency at the time ofinfection. Serial dilutions of a preparation of SIN replicon particlesexpressing a reporter gene were then diluted serially and used to infectthe cells in duplicate, at 37 C for 1 hour. Subsequently, the inoculumwas removed, the wells overlayed with agarose and the infected cellsincubated at 37 C. Plaques were visualized 48-72 hours later at whichtime they could be quantitated directly or after staining with a dyesuch as neutral red or crystal violet.

In the second instance, nucleic acid based detection of alphavirusreplicon particles as a means for quantitation was performed, using thebDNA amplification technique (Wilber, Immunol Invest 1997, 26:9-13) asone embodiment. FIG. 11 shows representative data from an experiment inwhich the titer of SIN replicon particles expressing HIV-gag antigen wasdetermined. A standard curve was developed initially using serialdilutions of SIN replicon particles expressing. GFP reporter, sinceprior quantitation of this material could be done by direct transfer ofexpression (TOE) assay and counting of green cells in a fluorescencemicroscope. As the vector replicon backbone was identical betweenSIN-GFP and SIN-gag, nucleic acid detection could then be done using anidentical nonstructural gene specific probe, since both particlepreparations differed only in the expressed heterologous gene.

In addition to quantifying the number of replicon particles in apreparation, it is also advantageous (or necessary) to determine thepresence or absence of contaminating replication-competent virus (RCV)in the preparation. Such RCV, if present, would have resulted from RNArecombination during the replicon packaging process. It has long beenrecognized by those of skill in the art that RCV testing may beperformed using standard plaque assay, with or without prior serialpassage in naive cultured cells. In order to increase the level ofsensitivity of RCV detection and to detect the multiple recombinationevents required for the generation of RCV in a “split helper” alphaviruspackaging system, a nucleic acid based assay as described herein hasbeen developed (FIG. 12). In this assay, a preparation containingreplicon particles is first extracted to isolate the nucleic acidsubstrate (e.g., RNA) present. The nucleic acid substrate is thenincluded in a first PCR reaction mixture comprising a firstoligonucleotide complementary to an alphavirus sequence not present inthe helper sequence(s) (e.g., nonstructural protein gene-specific,Rep-Fwd in FIG. 12), and a second oligonucleotide complementary to analphavirus structural protein gene (e.g., DH1 Rev or DH2 Rev, in FIG.12), wherein the structural protein is either a capsid protein or anon-capsid structural protein (e.g., glycoprotein). A reaction productfrom this reaction will specifically identify a recombination eventbetween the replicon vector and whichever structural protein genecontaining helper the second oligonucleotide was designed complementaryto. Thus, for example, if the second oligonucleotide was capsidgene-specific oligonucleotide DH2 Rev, a recombination event between thereplicon and the capsid gene-containing helper (e.g., DH2) could bedetected by the reaction product. In addition, based on length of thereaction product, multiple recombination events might also be detectedat this stage, but one could not necessarily ascertain whether suchrecombination events included all structural gene elements required forgeneration of RCV or simply recombination with multiple copies of thesame structural protein gene helper (e.g., two copies of capsid fromDH2).

Therefore, following amplification, the reaction product(s) from thefirst reaction mixture is included in a second PCR reaction mixturecomprising an oligonucleotide complementary to an alphavirus capsidprotein gene (e.g., DH2 Rev) and a oligonucleotide complementary to anon-capsid (e.g., glycoprotein) alphavirus structural protein gene(e.g., DH1 Fwd). For example, if the first reaction resulted in aproduct that was amplified using replicon and capsid specificoligonucleotides (e.g., Rep-Fwd and DH2 Rev), indicating recombinationbetween replicon RNA and capsid-containing helper RNA, then the abilityto synthesize a second reaction product in a second reaction containingthe first reaction product as template and oligonucleotidescomplementary to an alphavirus capsid gene (e.g., DH2 Rev) andnon-capsid structural protein gene (e.g., DH1 Fwd), would be indicativeof multiple recombination events.

Preferably, two separate first reactions are performed to identifyeither a capsid gene recombinant (e.g., using Rep-Fwd and DH2 Rev) or anon-capsid structural protein gene recombinant (e.g., using Rep-Fwd andDH1 Rev). Each of these first reactions then would be subjected to asecond reaction as described above, allowing for the identification ofall possible multiple recombination events that could result in RCV.

Similarly, such an approach may be used to identify packaging of adefective helper RNA into particles within a preparation, as well asco-packaging of replicon and defective helper RNA within particles. Forexample, the ability to amplify either capsid gene (e.g., DH2 Fwd plusDH2 Rev) or non-capsid structural protein gene (e.g., DH1 Fwd plus DH1Rev) sequences, without being able to amplify a product resulting fromrecombination (e.g., Rep-Fwd plus DH1 Rev or DH2 Rev) would beindicative of helper RNA present in packaged particles.

All references including publications, patent applications and patentscited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for purifying alphavirus replicon particles comprising: (a)contacting a preparation containing alphavirus replicon particles with atentacle ion exchange resin, under conditions and for a time sufficientto bind to said resin; (b) removing the portion of the preparation whichis not bound to said ion exchange resin from said ion exchange resin;(c) eluting the bound alphavirus replicon particles from said ionexchange resin; and (d) recovering said replicon particles.
 2. Themethod according to claim 1 wherein said tentacle ion exchange resin isa cationic exchange resin.
 3. The method according to claim 1 whereinsaid tentacle ion exchange resin is an anionic exchange resin.
 4. Amethod for purifying alphavirus replicon particles comprising: (a)contacting a preparation containing alphavirus replicon particles with afirst chromatography resin selected from the group consisting of atentacle ion exchange chromatography resin, a size exclusionchromatography resin, a hydrophobic interaction chromatography, and anaffinity chromatography resin; (b) recovering a first portion of thepreparation from the first resin, the first portion comprising thealphavirus replicon particles; (c) contacting the first portioncomprising the alphavirus replicon particles with a secondchromatography resin selected from the group consisting of a tentacleion exchange chromatography resin, a size exclusion chromatographyresin, a hydrophobic interaction chromatography, and an affinitychromatography resin; and (d) recovering a second portion from thesecond resin, the second portion comprising the alphavirus repliconparticles, thereby purifying alphavirus replicon particles.
 5. Themethod according to claim 4 wherein the first or second resin comprisesa tentacle ion exchange chromatography resin.
 6. The method according toclaim 4 wherein a first resin is a tentacle ion exchange chromatographyresin and a second resin is a size exclusion chromatography resin.
 7. Amethod for purifying alphavirus replicon particles comprising (a)contacting a preparation containing alphavirus replicon particles with afirst chromatography resin selected from the group consisting of an ionexchange chromatography resin, a size exclusion chromatography resin, ahydrophobic interaction chromatography, and an affinity chromatographyresin; (b) recovering a first portion of the preparation from the firstresin, the first portion comprising the alphavirus replicon particles;(c) contacting the first portion comprising the alphavirus repliconparticles with a second chromatography resin selected from the groupconsisting of an ion exchange chromatography resin, a hydrophobicinteraction chromatography, and an affinity chromatography resin; and(d) recovering a second portion from the second resin, the secondportion comprising the alphavirus replicon particles, thereby purifyingalphavirus replicon particles.
 8. A method for purifying alphavirusreplicon particles comprising: (a) contacting a preparation containingalphavirus replicon particles with a first chromatography resin selectedfrom the group consisting of a tentacle ion exchange chromatographyresin, a size exclusion chromatography resin, a hydrophobic interactionchromatography, and an affinity chromatography resin; (b) recovering afirst portion of the preparation from the first resin, the first portioncomprising the alphavirus replicon particles; (c) contacting the firstportion comprising the alphavirus replicon particles with a secondchromatography resin selected from the group consisting of an ionexchange chromatography resin, a size exclusion chromatography resin, ahydrophobic interaction chromatography, and an affinity chromatographyresin; and (d) recovering a second portion from the second resin, thesecond portion comprising the alphavirus replicon particles, therebypurifying alphavirus replicon particles.
 9. The method of claim 7wherein the alphavirus replicon particles are capable of expressing anantigen derived from a pathogenic agent.
 10. The method of claim 9wherein said pathogenic agent is selected from the group consisting ofviruses, bacteria, fungi, parasites, and cancerous cells.
 11. The methodof claim 7 wherein the alphavirus replicon particle preparationcomprises a therapeutic.
 13. The method of claim 7 wherein saidalphavirus replicon particle preparation expresses a lymphokine,cytokine, or chemokine.
 14. The method of claim 13 wherein saidlymphokine, cytokine or chemokine is selected from the group consistingof IL-2, IL-10, IL-12, gamma interferon, GM-CSF, macrophage inflammatoryprotein (MIP)3α, MIP3β, and secondary lymphoid tissue chemokine (SLC).15. A method for stimulating. an immune response within a warm-bloodedanimal, comprising administering to a warm-blooded animal a preparationof alphavirus replicon particles prepared according to claim
 7. 16. Themethod according to claim 15 wherein said alphavirus replicon particlepreparation expresses a lymphokine, cytokine, or chemokine.
 17. Themethod according to claim 16 wherein said lymphokine, cytokine orchemokine is selected from the group consisting of IL-2, IL-10-, IL-12,gamma interferon, GMCSF, macrophage inflammatory protein (MIP)3α, MIP3β,and secondary lymphoid tissue chemokine (SLC).
 18. A method of producingalphavirus replicon particles comprising: (a) infecting alphaviruspackaging cells with a seed stock of alphavirus replicon particles; (b)incubating the infected packaging cells in a bioreactor, underconditions and for a time sufficient to permit the production ofalphavirus replicon particles; and (c) harvesting culture supernatantscontaining said replicon particles.
 19. The method of claim 18 whereinsaid bioreactor is selected from the group consisting of an externalcomponent bioreactor, a suspension culture bioreactor, and a hollowfiber bioreactor.